The goal of this research is to positionally clone, identify by expression analyses, and determine cellular actions of genes on mouse Chromosomes (Chr) 1 and 4 that regulate bone mineral density (BMD). In clinical medicine, measurement of BMD establishes osteoporotic risk in susceptible individuals and predicts bone strength. Defining the relative contribution of various components of adult BMD, as well as the functional correlates of skeletal strength, remains a daunting task. The objective of this application is to exploit congenic sublines of mice carrying allelic differences for BMD quantitative trait loci (QTL) to uncover the gene(s) and cellular mechanisms regulating normal BMD. The inbred mouse strains providing the genetic variation being studied are C57BL/6J (B6;low BMD) and C3H/HeJ (C3H;high BMD) that differ significantly in peak femoral and vertebral BMD. The strongest, effective QTL in these strains, found on Chrs 1 and 4 have been genetically decomposed by congenic sublines and investigated for biological and gene expression. Chr 1 carries its BMD QTL in a 1.63 Mb region, while the original Chr 4 QTL region contains 3 QTL. Serum biochemical markers for BMD have not been informative, whereas cellular phenotypes in culture will be exploited in proposed studies. Hypotheses to be investigated are: 1) Chr 1 and 4 QTL region contains regulatory loci that can be mapped within a genomic interval region of ~1 cM or less and that candidate genes can be identified by gene expression differences;and, 2) each QTL for BMD will demonstrate corresponding changes in bone size, microstructure, and bone cell function. The specific aims are: 1) to genetically analyze the 1.63 Mb region of distal Chr 1 by bone gene expression in congenic sublines that do or do not carry BMD QTL, test the Ifi202b gene as a candidate locus, and test a 3-gene model for the Chr 4 QTL region;and 2) to functionally test osteoblast cultures for cell regulatory signal systems responding to QTL allelic differences. Methodologies to be employed include PCR genotyping of progeny from backcross-intercross matings to introduce genetic recombination into small QTL regions, quantitative real time PCR for gene expression, bone marrow cell cultures, primary osteoblast cultures, and reporter gene transfection of bone cells for intracellular signaling studies. The human health relevance for these studies derives from the fact that mouse distal Chromosomes 1 and 4 are homologous to human 1q22-24 and 1p36, which are genomic regions associated with human bone mineral density (BMD) regulation. Translation of results from mouse to human will facilitate finding candidate genes in human genome and in testing those candidates in mouse models.